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目的探讨遗传性出血性毛细血管扩张症发病的分子机制。方法 (1)用聚合酶链式反应-单链构像多态性分析(PCR-SSCP)寻找异常突变的外显子及其相邻剪接位点。(2)通过 DNA 测序确定突变的类型。(3)使用逆转录-聚合酶链反应(RT-PCR)方法确定剪接方式。结果用 PCR-SSCP 方法发现扩增 ALK1基因第4外显子及相邻的部分内含子片段有突变位点。对有突变该片段,用 DNA 测序检测,发现第4外显子的给位剪接点相邻碱基 A>T(IVS4+3 a>t)的突变,并用反向测序确认。用 RT-PCR 方法检测 ALK1基因表达 mRNA 情况,电泳和测序发现第4外显子的丢失。结论 ALK1基因的ⅣS4+3 a>t 突变,导致 ALK1基因表达异常,形成无功能截短蛋白,引起 HHT2。
Objective To investigate the molecular mechanism of hereditary hemorrhagic telangiectasia. Methods (1) Polymerase chain reaction-single strand conformation polymorphism analysis (PCR-SSCP) was used to search for abnormally mutated exons and their adjacent splice sites. (2) Determine the type of mutation by DNA sequencing. (3) The method of splicing was determined by reverse transcription-polymerase chain reaction (RT-PCR). Results PCR-SSCP method was used to amplify the ALK1 gene exon 4 and adjacent intron segments have mutation sites. Mutations in this fragment were detected by DNA sequencing and the mutation at the adjacent exon 4 to the putative splice junction of the exon 4 was found to be confirmed by reverse sequencing. The mRNA expression of ALK1 gene was detected by RT-PCR. The loss of exon 4 was detected by electrophoresis and sequencing. Conclusion The ⅣS4 + 3 a> t mutation of ALK1 gene leads to the abnormal expression of ALK1 gene, forming a nonfunctional truncated protein and causing HHT2.